The process was applied to the bonding of fused-silica glass and borosilicate glass, which is generally used in optical microscopy, and revealed higher bonding energy than fused-silica/fused-silica glass bonding. An annealing temperature lower than 200 °C was necessary to avoid crack generation by thermal stress due to the different thermal properties of the glasses. A fabricated micro/nanofluidic device exhibited a pressure resistance higher than 600 kPa. This work will contribute to the advancement of micro/nanofluidics.The bonding of glass substrates is necessary when constructing micro/nanofluidic devices for sealing micro- and nanochannels. Recently, a low-temperature glass bonding method utilizing surface activation with plasma was developed to realize micro/nanofluidic devices for various applications, but it still has issues for general use. Here, we propose a simple process of low-temperature glass bonding utilizing typical facilities available in clean rooms and applied it to the fabrication of micro/nanofluidic devices made of different glasses. In the process, the substrate surface was activated with oxygen plasma, and the glass substrates were placed in contact in a class ISO 5 clean room. The pre-bonded substrates were heated for annealing. We found an optimal concentration of oxygen plasma and achieved a bonding energy of 0.33–0.48 J/m The field of microfluidics has rapidly developed to realize various applications, including chemical analysis and synthesis, medical diagnosis, and tissue engineering ,2,3. RecFabrication methods for micro/nanofluidic devices made of glass have been reported. Micro- and nanochannels are usually fabricated on a glass substrate by wet etching and plasma etching, respectively, after the channel patterns are formed by lithography ,16. SurfIn order to solve these problems in bonding, recent studies have developed a method for bonding glass substrates by surface treatment with plasma and heating at low temperatures ,24. In tHowever, the low-temperature glass bonding method still has issues for general use. Adding fluorine atoms into oxygen plasma and heating the substrates while pressing with a controlled pressure requires the use of customized facilities in some cases. Furthermore, the bonding of different types of glass has not been achieved. Previous studies have verified the low-temperature bonding of fused-silica glass substrates ,24,25,28In the present study, we developed a low-temperature glass bonding process utilizing typical facilities available in clean rooms, and applied the process to fabrication of micro/nanofluidic devices. A bonding process with surface activation by oxygen plasma and without pressing substrates during the annealing was proposed. The oxygen plasma and heating temperature conditions were investigated, and the bonding of fused-silica glass substrates was verified by measuring the bonding energy. The verified bonding process was applied to the bonding of fused-silica and borosilicate glass substrates. The bonding energy of fused-silica/borosilicate glass substrates was evaluated. In addition, the pressure resistance of a micro/nanofluidic device made of fused-silica and borosilicate glass was evaluated by investigating liquid leakage from a nanochannel. This work provides a simple and easy low-temperature glass bonding process for the fabrication of micro/nanofluidic devices.10 Pa and 7.29 × 1010 Pa, respectively, according to the manufactures. The surface roughness of glass substrates was less than 0.3 nm.Fused-silica glass substrates with thicknesses of 0.25 and 0.70 mm  and borosilicate glass substrates with thicknesses of 0.17 and 0.25 mm  were prepared. The 0.17-mm borosilicate glass is used as a standard cover glass for microscopy. The Young’s modulus of fused-silica glass and borosilicate glass was 7.38 × 10For the confirmation of liquid leakage from nanochannels, a fluorescent aqueous solution, including 0.1-mmol/L fluorescein and phosphate-buffered saline was prepared.3 and SF6. To fabricate nanochannels of 10 to 100-μm width and 100 to 1000-nm depth, the channel pattern was formed by photolithography using THB-111N  as a photoresist. On the other hand, microchannels were fabricated by photolithography and dry etching. KMPR®1035  was used as a photoresist for micrometer-scale dry etching using a mixture of gaseous C3F8, CHF3 and Ar. Inlet holes with a diameter of 0.7 mm were produced on the substrate using a diamond-coated drill. After fabricating the channels and inlet holes, the device was constructed by the low-temperature bonding of the glass substrates as described in the following section.Micro/nanofluidic devices were fabricated by the top-down fabrication of micro- and nanochannels on glass substrates, based on reported methods ,30,31. NIn the present study, we propose a process of low-temperature glass bonding utilizing typical facilities available in clean rooms. To optimize the proposed bonding process, the effects of the pressure in the plasma reaction chamber and the heating temperature used for annealing on the bonding strength were investigated. The pressure in the reaction chamber was set to 40, 51 and 60 Pa by providing oxygen at a flow rate of 45, 70 and 100 mL/min, respectively, with evacuation by a vacuum pump. The heating temperature was set at 20 (room temperature), 50, 110, 200, 300 and 400 °C.L, was measured. The bonding energy, γ, between substrate 1 and substrate 2 is given by:bt is the thickness of the blade, st is the thickness of the glass substrate, and sE is Young’s modulus [The bonding strength of glass substrates was measured by a crack-opening test , which i modulus . As showThe pressure resistance of the micro/nanofluidic device was examined by observing the leakage of a fluorescein solution from a nanochannel. The fluorescence was observed by an inverted fluorescence microscope  combined with an objective lens , a 1.6× magnification lens, and a complementary metal-oxide-semiconductor (CMOS) camera . The pixel size of the CMOS camera was 5.86 μm, and the exposure time was set at 70 ms. The fluorescein solution was injected into the device by air pressure generated by a pressure controller .2 with heating temperatures ranging from 200 to 400 °C, which are comparable to that corresponding to a pressure resistance on the order of 100 kPa, as reported previously [In order to evaluate the bonding strength of the fused-silica glass substrates, we performed a crack-opening test. eviously ,24,25. TWe also investigated the effects of surface activation by oxygen plasma on the bonding energy. 2O → Si-OH + Si-OH, and the hydrophilicity of the surface increases. During the annealing after the pre-bonding, a dehydration reaction of silanol groups between the pair of substrates occurs to form covalent bonds of siloxane, according to Si-OH + Si-OH → Si-O-Si + H2O. As a result, the bonding of the substrates is achieved. Xu et al. [The results were evaluated to consider the mechanism involved in low-temperature glass bonding, which is not yet fully understood. Recently, Wang et al.  experimeu et al. ,24 and Ou et al.  indicateu et al. .At the optimized pressure in reaction chamber of 51 Pa, we further investigated the heating temperature for annealing. −6 K−1) and borosilicate (7.2 × 10−6 K−1). On the other hand, in the case of substrates with micro- and nanochannels , which is generally used for microscopic optical measurements. In the bonding process, using an annealing temperature lower than 200 °C was important to avoid crack generation by thermal stress due to the difference in thermal expansion coefficients between fused silica and borosilicate. At an annealing temperature of 110 °C, the bonding energy was around 0.5 J/cm2 and three times higher than that of fused-silica/fused silica glass bonding. The results suggest that the bonding energy depends on the glass material and increases in case of using borosilicate glass. We succeeded in device fabrication, and the fabricated micro/nanofluidic device had a pressure resistance higher than 600 kPa, which is sufficient for various micro/nanofluidic applications. In previous studies [2, which corresponds to pressure resistance of nanochannels on the order of 100–1000 kPa. In the present study, we developed conditions of low-temperature glass bonding utilizing oxygen plasma without fluorine atoms and verified bonding of fused-silica/fused-silica and fused-silica/borosilicate glass substrates. Bonding energy in similar order to that by previous studies was achieved. The results provide a simple and easy process of low-temperature glass bonding for the fabrication of micro/nanofluidic devices, enable the use of high-resolution optical microscopy designed for borosilicate cover glass, and will greatly contribute to advancements in micro/nanofluidics.We developed a simple process for low-temperature glass bonding utilizing typical facilities available in clean rooms and applied it to the fabrication of micro/nanofluidic devices by the bonding of fused-silica/fused-silica glass substrates and fused-silica/borosilicate glass substrates. The proposed bonding process was verified, and the bonding energy for fused-silica/fused-silica substrates at the annealing temperature of 200–400 °C was 0.33–0.48 J/m studies ,24,25, c


The microstructure was observed by scanning electron microscope (SEM). The 3D micro-CT and histological analysis were carried out to evaluate the bio-absorption of PR-DDM at 2 and 4 months. A smooth dentin collagen surface with symmetrical macro-pores and tube-type dentinal tubules (Ø 1–2 µm) with micro-cracks were observed on the perforated region. A significant number of custom-made macro-pores disappeared, and the size of the macro-pores became significantly wider at 4 months compared with the 2 months (p < 0.05) evaluated by 3D micro-CT. Histological images revealed the presence of multinucleated giant cells attached to the scalloped border of the PR-DDM. The morphological changes due to bio-absorption by the cellular phagocytes were comparable to the 3D micro-CT and histological images at 2 and 4 months. Altogether, the results demonstrated that the PR-DDM block was gradually absorbed by multinucleated giant cells and regenerated bone. Human PR-DDM might serve as a unique scaffold for extraoral bone regeneration.The aim of this study was to evaluate the bio-absorption and bone regeneration of human tooth-derived dentin scaffold, entitled as perforated root-demineralized dentin matrix (PR-DDM), after in vivo implantation into the critical-size iliac defects. The dentin scaffolds were prepared from human vital, non-functional teeth. Thirty artificial macro-pores (Ø 1 mm) were added after removing the enamel portion. The modified teeth were supersonically demineralized in 0.34 N HNO Vital human teeth provide an excellent source of stem cells, matrices, trace metal ions, and multiple growth factors, which are essential for bone tissue engineering. Dentin, a major component of a human tooth, is a bone-like mineralized tissue that shares a similar chemical composition with bone  . In addiA bone-inducing property of rabbit-derived DDM was discovered in 1967 ,14,15. THuman extracted non-functional teeth are routinely discarded as infective medical waste globally. Healthy teeth could be recycled to develop bone graft biomaterials that can overcome the limitations of autogenous bone and other bony substitutes ,21. An i®, DIA-BURS TF-11, Tochigi, Japan). A 1 mm area was cut from the apical portion using a diamond disk bur . To create an optimal pore size for bone ingrowth, a small round bur  was used to fabricate 1 mm hole, similar to in our previous study [®, Hwashin Tech. Co., Ltd., Seoul, Korea) in 5 L of 0.34 N HNO3 solutions at 100 W and 60 kHz for 30 min. The liquid temperature of the sample gradually increased with an increasing supersonic time up to 30 min, but they were maintained at less than 315 K to avoid denaturing proteins, as previously reported [n =9) were transformed into the PR-DDM, and the average length and width were calculated as 13.08 ± 0.92 mm and 8.41 ± 0.76 mm , the enamel and pulps were removed with a tapered fissure bur  was used to observe the surface texture, custom-made macro-pores, and native geometrical tunnel structures of the dentin. The scaffolds were fixed in 5% glutaraldehyde and 1% formaldehyde for 2 days. The PR-DDMs were dehydrated in a graded ethanol series  for 10–15 min and placed in vacuum desiccators for overnight drying. The following day, a total of five samples were sputter coated with 10 nm of gold and analyzed microscopically using a SEM at 1 mm and 5 μm magnifications (n = 5).The morphological characterization of the PR-DDM using a scanning electron microscopy (SEM)  and was performed according to the guidelines of the Declaration of Helsinki, and approved by the Health Sciences University of Hokkaido Animal Experimentation Regulation (No: 023), and Bioscience Department of Toya Laboratory of Hokudo Co., Ltd., Hokkaido, Japan. ®, Ethicon Inc., New Brunswick, NJ, USA) were used to secure the muscle flaps, and the skin incisions were closed with sterile 2-0 silk sutures. Animals were treated with intramuscular injections of penicillin G and dihydrostreptomycin (10 mg/kg) daily for 7 consecutive days. Adequate measures were taken to minimize the pain or discomfort to the experimental animals. At 2 months, three sheep and the remainder were humanely euthanized at 4 months post-operatively with intravenous injection of sodium pentobarbital-based euthanasia solution. The surgical graft sites were trimmed from the iliac crests after confirming the death of each animal and fixed in a 10% neutral phosphate-buffered formalin solution.The present study used six mature male Japanese Suffolk sheep (approximately 20–21 months old and 33–54 kg in weight). Intramuscular administration of atropine (0.06 mg/kg), xylazine hydrochloride (0.22 mg/kg), and ketamine (11 mg/kg) were used for general anesthesia. The anesthesia was maintained with endotracheal inhalation of 2% isoflurane throughout the operation. Under proper aseptic measures, a skin incision was made above the right iliac crest, and the tissues were dissected to expose the surgical site. Standardized critical-size bone defects (15 mm × 10 mm × 9 mm) in iliac crests were harvested by an oscillating saw under cooling with sterile physiologic saline, and PR-DDMs were grafted in the bone defects . After i®, Rigaku, Tokyo, Japan). Samples were imaged using following parameters: 90 kV tube voltage, 160 mA of automatic tube current, 0.12 mm slice thickness, and 17 s exposure time. For three-dimensional (3D) imaging data, we used the Osirix Image processing software (version 5.6) to compare the amount of PR-DDM bio-absorption at 2 months and 4 months post-implantation. Meanwhile, the number and size of artificial macro-pores in the PR-DDM were also analyzed with a 3D reconstructive volume rendering method using the same software.We evaluated the morphological changes due to bio-absorption and new bone regeneration of the PR-DDM at the bone defects using a micro-CT scanning device  and processed for paraffin embedding . Later, a 5 µm thickness of histological sections were prepared by a microtome  and stained with hematoxylin and eosin (HE) . The HE-stained sections were examined using an optical microscopy  for histological and histomorphometrical evaluation. p < 0.05 using Tukey’s test. A Windows computer with SPSS software ver. 19.0  was used to perform all statistical analysis.The statistical values are expressed as the mean . Each experiment was replicated at least three times. The one-way analysis of variance (ANOVA) was used to analyze statistically significant differences between the means of two groups. The statistical significance was accepted for The SEM of PR-DDM exhibited a homogenous dentin surface with symmetrical artificial macro-pores (1 mm in diameter) a. A highWe used an in vivo high-resolution 3D micro-CT to investigate and analyze the morphological changes and scaffold bio-absorption at 2 and 4 months. The difference in the PR-DDM bio-absorption was analyzed, and the images are presented in p < 0.05) (p < 0.05), indicating advanced dentin absorption  . The macsorption  Table 1p < 0.05 sorption .The present study described the dentin scaffold’s cellular absorption and graft stability with new bone formation in critical-size defects of adult sheep. The choice of the Japanese Suffolk sheep model provides a body weight, which is comparable to adult humans. In addition, the metabolic and bone remodeling rates of these sheep are similar to humans . CriticaScaffold-based bone tissue engineering requires definite geometrical features for the porous scaffold. An adequate porosity of suitable size and interconnections between the pores allows higher bone ingrowth than a non-porous scaffold ,36. A noMany bacteria live in the human oral cavity . As bactUntil now, the exact mechanisms of demineralized dentin degradation in the bone tissue have not been fully clarified. Both enzymatic digestion and cellular phagocytosis are the dynamic processes involved in the organ absorption ,42. The Biodegradable scaffolds play an increasingly important role in the bone regeneration process. It provides a bridge for new bone tissue growth into the scaffold-absorption areas and a platform for cells and growth factors to play a physiological role . Our preTo the best of our knowledge, this is the first study that shows the bio-absorption of the PR-DDM in the critical-size iliac defects in large animals. This study highlights a new medical technology that PR-DDM might contribute as a novel scaffold for bone engineering.3 demineralization into human teeth contributed to improve the 3D dense structure and the calcified surface into a porous and irregular type. Our study indicated that the PR-DDM block was gradually absorbed by multinucleated giant cells and regenerated bone as a biological scaffold. Owing to easy fabrication in a short period of time, human PR-DDM might be recycled as an innovative biomaterial for extraoral bone engineering.We successfully evaluated the bio-absorption of the PR-DDM block by 3D micro-CT and bone regeneration histologically in critical size iliac defects. The fabricated perforations and supersonically 0.34 N HNO


Schistosoma mansoni specific DNA detection in serum and urine using real-time polymerase chain reaction (PCR) in an endemic area before and after treatment.To detect acute schistosomiasis, low-intensity infections, or to verify the success of treatment with praziquantel, highly sensitive test methods are required. The aim of this study was therefore to demonstrate the performance of Schistosoma mansoni infection before and two times after treatment with praziquantel using serum- and urine based real-time PCR, point-of-care circulating cathodic antigen (POC-CCA) rapid diagnostic test and the microscopic Kato-Katz (KK) method. Kappa coefficient (κ) was used to estimate the agreement between these diagnostic tests compared to a combined “gold standard” of positive results by serum-based real-time PCR and/or positive egg counts determined by KK. Kendall’s Tau rank correlation was used to examine the relationship between cycle threshold (Ct)-values and egg counts and the Wilcoxon signed-rank test was used to compare the median Ct-values of the different examination time points.The study pursued a 1-week and 20-weeks longitudinal design with a treatment intervention among 36 study participants aged 18 to 70 years in the community of Kayenze, a fishing village in Ilemela district on the southern shore of Lake Victoria in north-western Tanzania between February and June 2018. Blood, urine and stool samples were collected from each participant to diagnose S. mansoni prevalence of 77.1% could be determined at baseline. In terms of sensitivity, serum-based real-time PCR (96.3%) and POC-CCA assay (77.8%) showed the highest results. The detection of DNA from urine samples showed the lowest sensitivity (33.3%). Treatment with praziquantel resulted in a significantly reduced prevalence of S. mansoni. No infection could be detected by Kato-Katz, with the POC-CCA test only 33.3%. The analysis of the median Ct values over time  showed that the Ct decreases significantly shortly after treatment (from 30.3 to 28) and increases above baseline level (34.9) three months later.By using the combined “gold standard” of the parasitological Kato-Katz test and/or serum-based real-time PCR, a S. mansoni DNA detection. However, as circulating DNA does not necessarily reflect the persistence of living worms in schistosomiasis, this method is less well suited to verify the success of treatment with praziquantel.The data presented here show that the serum-based real-time PCR exhibits excellent diagnostic accuracy, in contrast to the use of urine as sample material for  Schistosoma mansoni is one of the major species causing hepatic and intestinal schistosomiasis [S. mansoni eggs in stool and the urine rapid diagnostic test for detection of circulating cathodic antigens (CCA) are extensively used for screening of schistosomiasis [S. mansoni, the POC-CCA test has shown higher prevalence rates than the KK test [S. mansoni, which contains 121 bp tandem repeats and comprises at least 12% of S. mansoni genome of both sexes [S. mansoni DNA by real-time PCR in plasma also using this sequence [Human schistosomiasis is considered as one of the most prevalent parasitic infections in the world with an estimated 229 million people requiring preventive treatment . In sub-somiasis . The Katsomiasis . The KK somiasis . The poisomiasis . In nume KK test , 8. Neve KK test . False n KK test , 12. In  KK test , low-int KK test , 12. A s KK test . Thus, t KK test . The mos KK test . Based o KK test , real-ti KK test  and loop KK test  methods th sexes . Specifith sexes . Wichmansequence . Due to sequence . Therefosequence .In this study we investigated whether the detection of circulating free DNA from urine and serum samples using real-time PCR is suitable for diagnosing schistosomiasis compared to KK and POC-CCA test. In addition, we investigated whether serum-based real-time PCR can be used to assess the therapeutic success of praziquantel (PZQ) in a high prevalence setting on Lake Victoria. For this purpose, serum Ct values were compared during infection and after therapy.S. mansoni [S. mansoni infection high in adulthood [The study pursued a longitudinal design with a treatment intervention and was conducted between March and July 2018 in the community of Kayenze, a fishing village in Ilemela district on the southern shore of Lake Victoria in north-western Tanzania. The majority of the inhabitants depend on the lake for domestic and economic activities including fishing, farming, washing, bathing, cooking, drinking and recreation. The region is endemic for  mansoni –23 and tdulthood . Annual dulthood . The stuS. mansoni infection were assessed during a baseline survey and again 1 week (6–8 days) and 20 weeks (142–144 days) after PZQ administration (40 mg/kg). These participants belonged to a larger group selected for a prevalence study. The cohort examined for the current study consisted of 36 participants, who had been present at all follow-up time points and of whom serum and urine samples were available for real-time PCR analyses. The stool sample of one study participant was missing at baseline data collection. Serum samples were obtained after centrifugation of coagulated blood samples at 1200×g for 5 min. The supernatant was centrifuged for further 15 min at 3000×g and then stored frozen at -20 °C. The centrifugation steps were performed at room temperature. Aliquots of urine (15 ml) were preserved with Norgen® Urine Preservative  for nucleic acid extraction and stored at room temperature until use.Serum, urine and stool samples from adult participants with a proven S. mansoni eggs by the quantitative Kato Katz (KK) thick smear technique. For the KK method two thick smears were prepared from different parts of a single stool sample using a template of 41.7 mg , following a standard protocol [S. mansoni eggs by two experienced laboratory technicians of the National Institute for Medical Research (NIMR). For quality assurance, 10% of the negative and positive KK thick smears were re-examined by a third laboratory technician.Stool samples were evaluated for the presence of protocol , 26. AftSchistosoma by the POC-CCA cassette test according to the protocol and procedures described by the manufacturer . Trace readings were considered as positive test results.Urine samples were tested for CCA of DNA extraction from 2 ml serum and 4 ml preserved urine were performed using the QIAamp Circulating Nucleic Acid Kit according to the manufacturer’s suggestions . DNA was stored at − 20 °C after extraction.S. mansoni DNA samples was performed according to a previously published protocol [S. mansoni strain SM 1–7 (GenBank accession number M61098) described by Hamburger et al. [Detection of cell-free protocol  using a r et al. . Primer S. mansoni egg DNA) was positive and the exogenous Qiagen Internal Control to test successful amplification and to exclude the presence of PCR inhibitors was positive (Ct < 33) . A test was considered positive when the threshold was attained within 40 PCR cycles (Ct < 40).The 25 μl reaction mix contained 2.5 μl DNA, 1x QuantiFast Pathogen Master Mix , 400 nmol/L of each Sm Primer and 200 nmol/L of Sm probe. The PCR runs consisted of an initial step of 5 min at 95 °C followed by 40 successive cycles of 15 s at 95 °C and 30 s at 60 °C. The reaction was run on the StepOne real-time PCR system (Applied Biosystems). DNA detection was expressed by Ct - values. In every run, the non-template control was negative (Ct = 0), the positive control  and Microsoft Excel 2013 . A S. mansoni was determined for each diagnostic method and mean values  and medians were calculated for the Ct-values and eggs per gram stool (EPG). This study used a combined diagnostic “gold standard” of positive results by serum-based real-time PCR and/or positive egg counts as determined by KK. A combined “gold standard” is used to obtain a reliable result and has been described in other studies [The prevalence of  studies . Since tBy using the real-time PCR method, the Ct-value is displayed. The Ct is defined as the number of cycles required for the fluorescent signal to cross the threshold (i.e. exceeds background level). Ct levels are inversely proportional to the amount of target nucleic acid in the sample (the lower the Ct level the greater the amount of target nucleic acid in the sample). The relationship between Ct-values and egg counts was examined by the Kendall’s Tau rank correlation (τ). A nonparametric test (Wilcoxon signed-rank test) was used to compare the median Ct-values of the different examination time points.Study participants who provided sample material for the current study consisted of 28 females (77.8%) and 8 males with a mean age of 34 years (range: 18 to 70 years).S. mansoni was 34.3%. Egg loads varied between 12 and 1284 EPG with a median of 120 EPG. Real-time PCR detected S. mansoni DNA in 27 (75.0%) of serum samples with Ct-values ranging between 22.6 and 38 and a median Ct-value of 30.3. In urine, real-time PCR yielded 30.6% S. mansoni positive samples with Ct values between 33.9 and 40 and a median Ct-value of 38.5. The prevalence determined with the commercially available POC-CCA rapid test was 63.9%  of 34.8%. Kappa agreement between the microscopy and the defined standard was poor . The POC-CCA test had a higher sensitivity (77.8%) and NPV (53.9%) than the KK test. The Kappa statistic was 0.535 (P < 0.001). Serum-based real-time PCR displayed the highest sensitivity (96.3%) and NPV (88.9%), as well as high Kappa agreement . This technique missed one out of 27 positive cases. The lowest sensitivity (33.3%) and NPV (28%) was achieved by the method of urine-based real-time PCR. The Kappa agreement between this test and the “gold standard” was 0.119 (P = 0.25). These results are summarized in Table The “gold standard” used was a combination of the parasitological KK test and/or real-time PCR of serum samples. According to this procedure, 77.1% 27/35) 7/35 S. mS. mansoni after 20 weeks. Using the KK method, no infection could be detected (0%). The POC-CCA test detected 33.3% (12/36), the serum-based real-time PCR 58.3% S. mansoni positive samples . Twenty weeks after treatment, the median Ct-value increases significantly to 34.9 , and was significantly higher than baseline . The course of the Ct-values at the different examination time points is shown in Table P = 0.512) nor between the serum DNA levels and egg counts .Accordingly, serum-based real-time PCR showed the highest proportion of S. mansoni DNA in serum and urine was studied as an alternative diagnostic method to detecting eggs in stool or for detecting schistosome antigens in urine. The cell-free circulating schistosome DNA consists of fragments of parasite-derived DNA that is homogeneously distributed in plasma and other body fluids, such as urine or saliva of the host [S. mansoni by serum-based real-time PCR (75%), followed by the POC-CCA test (63.9%). Comparably few infections were identified with the urine-based real-time PCR method (30.6%). To determine the diagnostic accuracy, the combined results of microscopy and serum-based real-time PCR were used as reference in this study, with both methods being considered 100% specific. This procedure has also been reviewed and applied in other studies [S. mansoni infection is possible using urine as a source of DNA [In order to prevent egg-induced irreversible pathological reactions, highly sensitive tests for the diagnosis of schistosomiasis are of enormous importance. Hence, an ideal test should identify acute infections, low-intensity infections and residual infections, as well as allow assessment of treatment success and control programs. In this study, the value of detecting cell-free the host , 28–30.  studies , 31. Thee of DNA –35. In oe of DNA , 37.In addition, our results showed that the number of positives determined by serum-based real-time PCR was significantly higher 20 weeks after treatment than by parasitological stool examination. Using the KK method, no infection could be detected after treatment, as opposed to 58.3% with serum-based real-time PCR. There are several explanations for this discrepancy. First, this could be due to low sensitivity of the microscopy (44.4%) compared to the “gold standard” and could indicate that the egg-negatives were false negative. Second, reinfections with schistosomes are possible in high-transmission areas where humans are exposed to cercaria. The development from the schistosomula to the adult worm, which is capable of laying eggs, takes about 5–7 weeks , 38–40. S. mansoni DNA. The free DNA presumably originates from the adult worms killed by the therapy [S. japonicum by classical PCR showed that the DNA of this parasite was no longer detectable 6–8 weeks after treatment with praziquantel [S. mansoni-specific DNA is able to detect it from all stages of the life cycle. A positive PCR reaction shows that DNA of the parasite is present but not whether it comes from egg-producing worm pairs or, for example, larval stages [In the current study, we were able to detect changes in the Ct value after treatment with PZQ and thus draw conclusions about changes of the amount of free circulating  therapy , which l therapy , 42. Sin therapy , 43. Neviquantel . It is aiquantel , 46. OneDespite the fact that the results of the current study are based on a very small sample size and this may have affected the results, we conclude that serum-based real-time PCR has an added value in a variety of situations. Although it cannot replace the KK method or the POC-CCA test in endemic areas, this method is well suited to detect schistosomiasis very early or infections with low intensities, because of its high diagnostic accuracy. This is particularly useful for travelers and migrants to prevent possible chronic complications of an unrecognized disease or for long-term monitoring of control interventions. However, as circulating DNA does not necessarily reflect the persistence of living worms in schistosomiasis, this method is less well suited to verify the success of treatment with praziquantel.Additional file 1. Dataset


This study proposes a cascaded network model for generating high-resolution doses  from low-resolution doses  with reduced computation time.95%, V70%, V50%, and V30%) for the low-resolution and predicted high-resolution doses relative to the baseline high-resolution dose. Gamma analysis (between the baseline dose and the low-resolution dose/predicted high-resolution dose) was performed with a 2%/2 mm criterion and 10% threshold.Using the anisotropic analytical algorithm with three grid sizes  and the Acuros XB algorithm with two grid sizes (1 and 3 mm), dose distributions were calculated for volumetric modulated arc therapy plans for 73 prostate cancer patients. Our cascaded network model consisted of a hierarchically densely connected U-net (HD U-net) and a residual dense network (RDN), which were trained separately following a two-dimensional slice-by-slice procedure. The first network (HD U-net) predicted the downsampled high-resolution dose (generated through bicubic downsampling of the baseline high-resolution dose) using the low-resolution dose; subsequently, the second network (RDN) predicted the high-resolution dose from the output of the first network. Further, the predicted high-resolution dose was converted to its absolute value. We quantified the network performance using the spatial/dosimetric parameters  between the baseline and predicted doses were observed. The gamma passing rates for the predicted high-resolution does were higher than those for the low-resolution doses.The proposed model accurately predicted high-resolution doses for the same dose calculation algorithm. Our model uses only dose data as the input without additional data, which provides advantages of convenience to user over other dose super-resolution methods. Precises (OARs) . In genes (OARs) , the anis (OARs) , 9, Acurs (OARs) , 11, ands (OARs) , in commvia the IMRT plan because of small OARs involved, for example, optic nerve and cochlea. Chow et al.  With film/Monte Carlo evaluations, Gagne et al. -19 With  reportedw et al. , 19 showAs reported in aforementioned studies, a 1 mm grid facilitates precise dose calculation by reducing the volume averaging effect , 21, whiDespite the dosimetric advantage associated with using a 1 mm grid, many clinics typically use 2–5 mm grids because of relatively long computation time for a 1 mm grid. We reported the computation times for the AAA and the AXB algorithm according to grid sizes in prostate VMAT plans  in a previous study  have helped solve complex problems in radiotherapy , such asTo overcome these limitations, we propose a cascaded network model capable of generating high-resolution doses (with a 1 mm grid) from low-resolution doses (with ≥3 mm grids) with reduced computation time in prostate VMAT. This paper describes the details of network construction/data preparation /network training and quantifies the network performance by calculating the spatial/dosimetric parameters for the low-resolution and predicted high-resolution doses relative to the baseline high-resolution dose with a 1 mm grid.Our cascaded network model consisted of (a) a hierarchically densely connected U-net (HD U-net) , which uvia each convolution operation) and an activation function were modified to 24 and a leaky rectified linear unit (ReLU), respectively. In general, the original HD U-net (The HD U-net predicts the downsampled high-resolution dose (derived from the baseline dose with a 1 mm grid) using the low-resolution dose as the input. Therefore, the network was trained using low-resolution (input) and downsampled high-resolution dose (label) datasets. HD U-net  except fvia skip connection between the output of the first convolved layer and the output of the last layer (before upsampling). The ESPCN, which performed upsampling via rearrangement of the pixels of feature maps, was applied to the upsampling layer. At the last layer, a 3×3 convolution (stride 1 and filter 1) without activation function was used to map 32 features to 1 feature.We used the RDN structure for image super-resolution proposed by Zhang et al. . The RDNData preparation consisted of acquisition, preprocessing , data selection, and augmentation steps. For the data acquisition, 73 patients who received prostate VMAT (prescription: 78 Gy/39 fractions for 20 patients and 70 Gy/28 fractions for 53 patients), were selected. For each patient, dose distributions were computed according to calculation algorithms and grid sizes under the same treatment plan; the high-resolution doses  and low-resolution doses  were acquired. Subsequently, downsampled high-resolution doses were generated by bicubic interpolation of the high-resolution doses to a matrix size of the low-resolution doses. The datasets were split uniformly at random into training (80%) and test groups (20%). In preprocessing, the doses for test/training datasets were normalized according to the prescription dose to correct for bias deriving from different prescription doses (78 or 70 Gy). Then, cropping was used to remove unnecessary regions such as those outside of the body structure. In the data selection step, only axial dose planes having a pixel dose with >10% of the prescription  were selected. This data selection process was not applied to the test dataset used to quantify the network performance. Finally, the training dataset was augmented using flip and rotations .2. These CT images were exported to Eclipse TPS . Then, contours for PTV and OARs  were delineated by a radiation oncologist. Subsequently, prostate VMAT plans were generated with prescription doses of 78 Gy in 39 fractions (53 patients) or 70 Gy in 28 fractions (20 patients). The details of the plan information, including dose volume constraint, were described in our previous study  of our institute approved the data collection . Computed tomography images were acquired for 73 prostate cancer patients using a CT scanner . The pixel spacing of the acquired CT images was 1.17×1.17 mmus study . For eacMaterials and Methods. A.). For training of the AAA 5 model, 64×80 dose planes were paired with 320×400 dose planes.The preprocessing step comprised normalization and cropping. First, the datasets were normalized according to the prescription dose to correct for bias derived from the two different prescriptions . Cropping was used to remove irrelevant regions such as those outside of the body structure, and was performed to fit the following matrix sizes: (a) 96×128 for a 3 mm grid, (b) 64×80 for a 5 mm grid, (c) 288×384 for a 1 mm grid, and (d) 320×400 for a 1 mm grid. The 96×128 and 64×80 sizes indicated the matrix dimensions for axial dose planes using 3 and 5 mm grids, respectively. The other sizes represented the matrix dimensions of axial dose planes in the high-resolution doses. The 96×128 and 288×384 dose planes were paired together for training of the AAA 3/AXB 3 models . This is because, for each patient, most dose planes did not include a sharp dose gradient region near a PTV structure even though the effect of grid size on dose accuracy would be relevant to this region , 39. ThiMaterials and Methods. A.), and the trained models were evaluated using the test dataset. The training-evaluation cycle was repeated 5 times using a five-fold cross validation technique. We averaged the outcome from these five repetitions.Our proposed cascaded networks (HD U-net and RDN) were trained separately using training dataset. The test dataset produced no effects, such as network hyperparameter tuning, during the training process. The HD U-net (first network) was trained with the low-resolution doses (input) and downsampled high-resolution doses (label) for the same dose calculation algorithm. After the downsampled high-resolution dose (input) and the corresponding high-resolution dose planes (label) were converted to percentages, the RDN (second network) was trained using these data regardless of the dose calculation algorithm. We trained three models . Prior to the training the RDN (second network), the downsampled high-resolution dose (input) and the corresponding high-resolution dose planes (label) were converted to percentages. Then, the RDN was trained using these data, regardless of the dose calculation algorithm. The initialization and optimization of the network parameters were performed following the same procedure as for the HD U-net. The implementations were conducted on a desktop computer (NDVIA Geforce GTX 1050 Ti 4GB) using a framework of TensorFlow 2.0.0  separately. To train the HD U-net (the first network in the cascaded networks), the low-resolution dose planes (input) and the corresponding downsampled high-resolution dose planes (label) for the same dose calculation algorithm were used. We defined the loss function as the mean square error between the predicted dose (network output) and the label dose planes. At the start of the training process, trainable parameters for the network were initialized by the Glorot uniform initializer . The netow 2.0.0 . This trDose distribution differences were generated by subtracting the baseline high-resolution doses from the low-resolution doses (which were linearly interpolated to a matrix size consistent with the baseline) and the predicted high-resolution doses, respectively. In addition, to quantify the accuracy, the DSCs for the isodose volume between the baseline and low-resolution doses/predicted doses were calculated according to the following equation:where A and B denote the binary volumes by threshold (≥ arbitrary isodose) for the baseline and other doses, respectively. We plotted the DSC curves ranging from 0 to 100% isodose volumes.To quantify the dosimetric accuracy, dose volume histograms for the PTV and OARs  were calculated for the baseline high-resolution, predicted high-resolution, and low-resolution doses.via the following equations:For the PTV, dosimetric parameters, such as the mean dose, maximum dose, minimum dose, and the V95% (the ratio of the percentage of volume irradiated by 95% or more of the prescription dose to the PTV), were calculated. Furthermore, the HI (describing the uniformity of the dose distribution within the target) and CI (the ratio of the reference isodose volume to the PTV) were calculated where D2, D98, and D50 denote the doses covering 2%, 98%, and 50% of the PTV, respectively, andRI denotes the reference isodose volume (set at 95%) for the body, and TV represents the physical volume of the PTV.where Vn%, which is the ratio of the volume irradiated by n% or more of the prescription dose to the OAR volume , were evaluated. For left/right femoral heads, mean, maximum, and minimum doses were analyzed.For the rectum/bladder, mean, maximum, and minimum doses as well as VThree-dimensional gamma evaluation was performed to quantitatively compared the baseline high-resolution dose and low-resolution dose/predicted high-resolution dose in the PTV, rectum, bladder. The gamma passing rate was acquired under a dose difference/distance-to-agreement acceptance criterion (2%/2 mm) for global normalization. A low dose threshold was set to 10% of maximum dose .Statistical analyses were performed to evaluate the statistical significance between the baseline high-resolution doses and the low-resolution/predicted high-resolution doses for the dosimetric parameters. In addition, gamma passing rate for low-resolution dose was statistically compared with that for predicted high-resolution dose. The paired t-test or the Wilcoxon signed-rank test was performed using SPSS Statistics 21  after a normality check using the Shapiro-Wilk test. The statistical significance was decided as p-value of <0.05.This section describes the results for the PTV, bladder, and rectum; the results for other OARs are described in the The mean computation times to predict the high-resolution dose for an axial 2D dose plane were 0.017 s and 0.011 s for the AAA 3/AXB 3 models (from 3 to 1 mm grid) and the AAA 5 model (from 5 to 1 mm grid), respectively.95%, and CI for the PTV in the low-resolution doses were underestimated compared to those from the baseline dose. The minimum dose and HI were overestimated. The differences in the dosimetric parameters between the baseline high-resolution dose and the low-resolution dose with a 3 mm grid were 0.3 Gy (mean dose), 2.3 Gy (max. dose), −0.2 Gy (min. dose), −1.0% (V95%), −0.01 (HI), and −0.04 (CI), respectively. The corresponding parameter differences for a 5 mm grid were 0.7 Gy (mean dose), 3.5 Gy (max. dose), −0.6 Gy (min. dose), −5.2% (V95%), −0.03 (HI), and −0.11 (CI), respectively. By contrast, the corresponding differences for the predicted high-resolution doses for the AAA 3 and AAA 5 models were reduced. These results illustrate that the predicted doses showed a closer agreement with the baseline dose compared to the low-resolution doses. For both the bladder and rectum, the majority of the parameters from the predicted doses demonstrated closer agreement with the baseline doses compared to the low-resolution doses, with similar results observed in the other OARs .Among the dosimetric parameters for the AXB dose Table 2,70% for the bladder/rectum). By contrast, no significant differences  were observed between the baseline and the predicted high-resolution dose (AAA 3 model) for the majority of parameters (Comparing the baseline AAA doses (with a 1 mm grid) with the low-resolution AAA doses (with a 3 mm grid), the p-values for most parameters were less than 0.01. This indicates that there are statistically significant differences between the two doses except for a few parameters , which were trained separately. The proposed model took <0.02 s to predict one axial high-resolution dose plane from the low-resolution dose plane (with 3 or 5 mm grids) for the same dose calculation algorithm. Compared to low-resolution doses, the predicted high-resolution doses were visually similar to the baseline high-resolution doses; furthermore, the DSC values of the predicted doses were closer to 1 than those of the low-resolution doses. The DVH curves for the PTV/OARs for the predicted dose were more consistent with the DVH curves for the baseline than the low-resolution dose. For the predicted dose, the average of the dosimetric parameters, including mean dose, maximum dose, minimum dose, HI, CI, V95%, V70%, V50%, and V30% were closer to corresponding parameters obtained in the baseline than the low-resolution dose. Moreover, the majority of these parameters demonstrated no statistically significant differences between the baseline and predicted doses. The gamma passing rates based on a 2%/2 mm criterion for the predicted high-resolution does were higher than those for the low-resolution doses. These results indicate that the proposed network is capable of achieving dose super-resolution with reduced computation time.The AAA 5 model (prediction of the high-resolution AAA dose from the low-resolution AAA dose with a 5 mm grid) showed poor performance when compared to the AAA 3 model (prediction from the low-resolution AAA dose with a 3 mm grid). One reason for this is that the quality of the input for the AAA 5 model was relatively poor compared to the AAA 3 model. Another reason is the large super-resolution scale (×5 for the AAA 5 model vs. ×3 for the AAA 3 model), which can cause a blurring effect in the predicted dose. Similar observations were recorded by previous studies exploring image super-resolution in the field of computer vision , 44, 45.Our study focused on achieving dose super-resolution for the same dose calculation algorithm, as opposed to the conversion of the AAA-calculated doses to the AXB-calculated doses. Obviously, converting the AAA-calculated dose with a 5 mm grid to that for the AXB algorithm with a 1 mm grid has a great advantage for computation time; this is because the AAA with a 5 mm grid requires the shortest time for dose calculation, whereas the AXB algorithm with a 1 mm grid requires the longest time . HoweveOur proposed method combined some advantages of previously reported networks , 38, 47.Although this study focused on prostate VMAT plans only, retraining the network will likely enable the proposed method to be applied to other sites, such as the lungs. For example, a previous study related to lung VMAT recommended using the AXB algorithm with a 2.5 mm grid instead of a 1 mm grid due to clinically acceptable calculation time despite the higher accuracy offered by the 1 mm grid . Our metOur dose super-resolution method can be applied in clinic as a useful tool to assist in improvement of treatment planning. For example, patient cases with noticeable errors in dose calculations by large grid sizes can be quickly identified through subtraction between calculated low-resolution dose and predicted high-resolution dose. For these cases only, the dose distributions can be re-calculated with a 1 mm grid. In other words, our method can provide the radiotherapy staff to information on cases that require dose calculation with a small grid size.The proposed method has some limitations. One of these limitations is the small dataset (73 patients). To mitigate the effects (such as overfitting) relating to the small dataset, we used k-fold cross validation (set at k = 5) and data augmentation (flip and rotations), which are widely recognized as appropriate techniques for reducing overfitting , 50. FurWe propose a cascaded network model that exhibits performance similar to the dose super-resolution of a 1 mm grid with reduced computation time in prostate VMAT. Our findings indicate a good agreement between baseline high-resolution doses with a 1 mm grid and predicted high-resolution doses. Our dose super-resolution approach uses only dose distributions without additional data such as CT images, which provides advantages of convenience to the user over other dose super-resolution methods. Therefore, our model could be easily applied to the clinic. The proposed method demonstrates immense potential and can be extended easily to other sites, such as the lungs, through retraining the network. In addition, our model could be a useful tool in other radiotherapy technique such as adaptive radiation therapy.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Seoul National Bundang Hospital Institutional Review Board Protocol (B-2004/608-112) for retrospective 73 cases. The patients/participants provided their written informed consent to participate in this study.Study concept and design: D-SS, TS, and J-BC. Data acquisition: D-SS, K-HK, J-SK, and J-BC. Quality control of data: S-WK, S-HK, and J-SK. Network construction and training: D-SS, K-HK, and S-WK. Data analysis and interpretation: D-SS, K-HK, S-WK, S-HK, T-HK, D-SK, WC, TS, and J-BC. Manuscript preparation and editing: D-SS and J-BC. All authors contributed to the article and approved the submitted version.This research was supported by the Mid-career Researcher Program (Grant No. 2018R1A2B2005343) through the National Research Foundation of Korea and by the Seoul National University Bundang Hospital (SNUBH) Research Fund (Grant No. 13-2018-013).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In diabetic subjects, Malays had significantly higher RAGE levels compared to Chinese and Indians . Low α-tocopherol level  and high insulin resistance  were linked strongly to the development of pre-diabetes. Low β-cell function  and high RAGE level  were linked strongly to the development of diabetes from pre-diabetes. These factors might be involved in the development of diabetes, along with genetic and environmental factors.Malaysian national morbidity surveys on diabetic prevalence have shown ethnical variation among prediabetic and diabetic populations. In our attempt to understand this variation, we studied the α-tocopherol, insulin resistance, β-cell function and receptor of advanced glycation end-products (RAGE) levels, as risk factors of type 2 diabetes, among the different ethnicities. In total, 299 subjects of Malay, Chinese, Indian and aboriginal Orang Asli (OA) heritage were recruited from urban and rural areas of Malaysia by stratified random sampling. Serum α-tocopherol concentrations were measured using high performance liquid chromatography (HPLC) and insulin concentrations were measured using enzyme-linked immunosorbent assay (ELISA). In subjects with pre-diabetes, OAs had the highest α-tocopherol level, followed by Chinese and Malays (0.8938, 0.8564 and 0.6948 respectively;  Type 2 diabetes has become a major public health problem globally. According to the World Health Organization WHO, 20140), approximately 422 million adults aged 18 years and older worldwide have diabetes, and the global prevalence has almost doubled from 4.7% in 1980 to 8.5% in 2014 [40, approThe prevalence of diabetes is also increasing rapidly. The Malaysia National Health and Morbidity Survey (NHMS) 2015 showed the prevalence of diabetes increased from 11.6% in 2006 to 17.5% in 2015 and to 18.3% in 2019 . The preIn Malaysia, Indians have the highest diabetes prevalence (22.1%), followed by Malays 14.6%), Chinese (12%) and indigenous people (2%) . In Sing%, ChinesThe indigenous people of Malaysia, the Orang Asli , comprise only 0.76% of the population in Peninsular Malaysia .Type 2 diabetes is a progressive endocrine disease caused by a combination of failure of the β-cells of the pancreas to secrete enough insulin and the failure of the body to utilize insulin effectively . In respRecent studies have shown the receptor for advanced glycosylation end products (RAGE) to be directly involved in the pathogenesis of diabetes. Lee et al. found that RAGE and its ligands induced the apoptotic death of human islet cells and INS-1 cells (rat pancreatic β-cell line) . RAGE knp = 0.0004) [p < 0.01) [Vitamin E occurs naturally as tocopherols and tocotrienols, each with α-, β-, γ- and δ- isoforms . Studies 0.0004) . The Ins < 0.01) . Several < 0.01) ,22, whic < 0.01) .This study aims to determine the α-tocopherol, insulin resistance, β-cell function and RAGE levels, as risk factors of type 2 diabetes, and compare these levels among the Malay, Chinese, Indian and OA subjects, associating it with the stage of diabetic development, i.e., non-diabetes, pre-diabetes and diabetes.https://www.openepi.com/Menu/OE_Menu.htm [This is a cross-sectional study of 299 subjects recruited in 2018/2019. The sample size calculation was based on a two-sided confidence interval of 95%, power of 80% and ratio of sample size of 1:1 to detect a mean difference of serum-corrected α-tocopherol concentration between non-diabetic  and diabetic subjects , as seen in the Finnish Study [Menu.htm .Non-diabetic, pre-diabetic and diabetic subjects were recruited from community health screening programs conducted in the Johor Bahru, Masai and Kulai districts of Johor, Malaysia, as well as from the Clinical Research Centers of Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia in Bandar Sunway, Selangor state. The definition of type 2 diabetes status was based on the Australian Type 2 Diabetes Guidelines ,26—the nThe inclusion criteria were as follows: (1) age > 18 years old; (2) consent given; (3) more than 500μL of stored sera being available; (4) data on demographics, anthropometric measurements, HbA1c, fasting blood glucose and lipid profile being available; (5) not on vitamin E supplements for the past 6 months; (6) not having any microvascular or macrovascular diabetic complications.Within the non-diabetic, pre-diabetic and diabetic groups, the subjects were subdivided into subgroups of comparable numbers of subjects based on their ethnicities, as follows: Malay, Chinese, Indian and OA. The study was conducted in accordance with the Declaration of Helsinki, with extended approval obtained from Monash University Human Research Ethics Committee (Project number: 18149). Informed consent was obtained during community health screening clinics.Sample sera were stored in identity marked Eppendorf tubes at −80 °C for ~2 years, and the α-tocopherol samples were stable kept at −80 °C for ~7 years .The extraction of analytes from serum samples was published previously . In totaHPLC was used to quantify the α-tocopherol concentration of samples. Purified sera extract was injected onto a Poroshell 120 EC-C18 2.1 × 100 mm column, with a 2.7 μm particle size. The mobile phase containing a mixture of acetonitrile, tetrahydrofuran, methanol and 1% ammonium acetate solution at 68:22:7:3 was eluted at a flow rate of 0.3 mL/min, using the Agilent 1200 Series Reverse Phase HPLC System  containing a G1312A binary pump in isocratic mode, a G1365C MWD detector (at 300 nm), and a programmable G1367D HiP sampler with a temperature-controlled sample tray. Agilent Chemstation Version B.04.03-SP2 was used for peak integration, sample calibration and data analysis. For the analysis of serum α-tocopherol concentration, a 6-level calibration curve was constructed using α-tocopherol standards of concentrations ranging from 15 μmol/L to 90 μmol/L. An external standard with a known concentration of α-tocopherol was included in all runs. The sera extract was injected alternately with a mobile phase-containing blank sample. The degree of analytical background response of α-tocopherol in serum  was assessed by calculating the mean and standard deviations of the response of 212 blank samples; the detection limit was 2.113895 μmol/L (σ: 1.644104).Enzyme-linked immunosorbent assay (ELISA) kits were used to measure serum insulin levels  and serum RAGE levels  based on the manufacturer’s protocol. Measurements were done in duplicates and the optical density was measured using a BioTek EON High Performance Microplate Spectrophotometer  at 450 nm. The intraassay and interassay coefficients of variation were 5.0% and 4.5%, respectively, for the human insulin kits, and were 5.0% and 5.8%, respectively, for the human RAGE kits.The fasting serum insulin and blood glucose levels were used in the updated homeostatic model assessment (HOMA2) calculator, to compute the insulin resistance (HOMA2 IR) and β-cell function (HOMA2%B) .Statistical Package for Social Sciences (SPSS) version 25  was used to conduct statistical analysis of data. As lipophilic molecules such as vitamin E are transported intravascularly along with lipids in the form of lipoproteins, serum lipid levels can affect serum α-tocopherol concentration . Therefoadj: lipid-adjusted ɑ-tocopherol level; y: measured ɑ-tocopherol level; B1: regression coefficient for total cholesterol; x1: measured total cholesterol; x1,0: standard value of total cholesterol set at 5.18 mmol/L; B2: regression coefficient for triglyceride; x2: measured triglyceride; x2,0: standard value of triglyceride set at 1.25 mmol/L. The standard values of total cholesterol and triglyceride were chosen respectively as values that correspond to a low cardiovascular risk in the study population, as stated in the Jordan et al. study [Yl. study . The lipThe data were tested for normality. A non-parametric Kruskal–Wallis test was used to compare the levels of serum α-tocopherol, HOMA2 IR, HOMA2%B and RAGE between the following: (i) non-diabetic, pre-diabetic and diabetic study subjects; (ii) non-diabetic, pre-diabetic and diabetic subjects in subgroups of Malay, Chinese, Indian and OA ethnicity; (iii) Malay, Chinese, Indian and OA subjects in subgroups of non-diabetes, pre-diabetes and diabetes. The data were presented in medians with interquartile range (IQR). Post hoc Dunn’s test was then used to pinpoint specific medians that showed significant difference from the others. Binary logistic regression was used to assess the association of corrected serum α-tocopherol level, HOMA2 IR, HOMA2%B, and serum RAGE concentration with the development of diabetes. The lipid-corrected α-tocopherol concentration, HOMA2 IR, HOMA2%B and serum RAGE concentration were dichotomized using the median as a cut off to maximize statistical power. Additionally, Spearman’s correlation was used to examine the correlation of corrected serum α-tocopherol concentration with HOMA2 IR, HOMA2%B and serum RAGE concentration.The median serum α-tocopherol and corrected serum α-tocopherol concentrations, HOMA2 IR, HOMA2%B and serum RAGE concentrations in the study population, grouped as non-diabetics, pre-diabetics and diabetics, are outlined in p = 0.185). However, when serum α-tocopherol concentration was corrected based on lipid levels, a significant difference was observed (p = 0.008). Amongst the groups, non-diabetics had the highest corrected serum α-tocopherol concentration . Pre-diabetics had a 21% lower corrected serum α-tocopherol concentration  compared to the non-diabetics. Diabetics had a higher corrected serum α-tocopherol concentration  than the pre-diabetics, albeit lower compared to the non-diabetics.In the overall study population, there was no significant difference in serum α-tocopherol concentration between the population groups (p = 0.007) and in β-cell function (p < 0.0000001) amongst the groups. Pre-diabetics  had double the insulin resistance of non-diabetics . Diabetics  had a higher insulin resistance than the non-diabetics, but lower compared to pre-diabetics. β-cell function was the highest in pre-diabetic subjects . This was followed by that of non-diabetic subjects . β-cell function in diabetic subjects  was the lowest amongst the groups, and was half that of the non-diabetic subjects and one-third that of the pre-diabetic subjects.Significant difference was observed in insulin resistance (p < 0.001).Serum RAGE concentration was the highest in diabetic subjects  followed by pre-diabetic subjects  and non-diabetic subjects ; the difference seen was statistically significant (p = 0.003). The pattern was akin to that of the overall study population, where the corrected serum α-tocopherol concentration was the highest in non-diabetics  and lowest in pre-diabetes , and in diabetes  the level was higher than in pre-diabetics but lower than in non-diabetics. There was also a significant difference in insulin resistance in the Malays across the diabetic stages (p = 0.038). The insulin resistance increased in an ascending order , pre-diabetics  and diabetics ), with the level being almost ten times higher in diabetics compared to non-diabetics. Across all diabetic stages, insulin resistance amongst the Malays was significantly lower compared to the overall study population (p = 0.0003). The serum RAGE concentration in the diabetic Malays  was significantly higher than in the non-diabetics  and pre-diabetics . This was distinct compared to the overall population, where the RAGE levels were similar in non-diabetics and prediabetics and the increase in RAGE levels in overall diabetics was not as pronounced as that seen in the Malays . This pattern was similar to the overall population where insulin resistance was highest among the pre-diabetics . The main difference was that the insulin resistance was the lowest in diabetics . In non-diabetics , the level was higher than in the diabetics but lower than in the pre-diabetics. Notably, insulin resistance in the Chinese was significantly higher than that in the overall population in the non-diabetics and prediabetics groups, but not in the diabetic group. (p < 0.0000001). Corresponding to the insulin resistance, the β-cell function was also the highest in pre-diabetics , followed by non-diabetics  and diabetes . The β-cell function of the Chinese was higher than the overall population across all diabetic stages . In the present study, a comparable sample size could not be obtained for pre-diabetic Indians. The β-cell function of diabetic Indians  was more than one-third that of the non-diabetic Indians .In the Indians, a significant difference was observed in the β-cell function in non-diabetics and diabetics (p = 0.00003) and pre-diabetic (p = 0.023) Malay, Chinese, Indian and OA subjects. In non-diabetes, the Chinese  had the highest serum α-tocopherol concentration, followed by the Malays , the Indians  and the OAs . Notably, in non-diabetics, the serum α-tocopherol concentration in the Chinese was almost double that of the OAs. The trend was similar in pre-diabetes, where the Chinese  had the highest serum α-tocopherol concentration, followed by the Malays  and the OAs . However, no significant difference was seen in the serum α-tocopherol concentrations of diabetic Malay, Chinese, Indian and OA subjects.Significant difference was seen in the serum α-tocopherol concentration amongst the non-diabetic (p = 0.023). In pre-diabetes, the OAs  had the highest corrected serum α-tocopherol concentration instead, followed by the Chinese  and the Malays . In pre-diabetes, the Malays had corrected serum α-tocopherol concentrations lower than the overall study population, while the Chinese and the OAs had levels higher than the overall study population. When the serum α-tocopherol concentration was lipid-corrected, a significant difference could only be seen in the pre-diabetic Malay, Chinese and OA subjects (p = 0.003) and pre-diabetic (p = 0.005) Malay, Chinese, Indian and OA subjects. In non-diabetes, the Chinese  had the highest insulin resistance, followed by the Indians , the OAs  and the Malays . In non-diabetes, the insulin resistance of Chinese subjects was double that of the OAs and ten times that of the Malays. This trend continued in pre-diabetes, where the Chinese  had the highest insulin resistance, followed by the OAs  and the Malays . In pre-diabetes, the insulin resistance of the Chinese was triple that of the OAs and five times that of the Malays. There was no significant difference in the insulin resistance of diabetic Malay, Chinese, Indian and OA subjects.There was a significant difference in insulin resistance amongst non-diabetic (p = 0.00006) and pre-diabetes (p = 0.0002). In non-diabetes, the Chinese  had the highest β-cell function, followed by the Indians , the OAs  and the Malays . In non-diabetes, the Chinese had β-cell functions four times that of the OAs and more than eight times that of the Malays. In pre-diabetes, the Chinese  had the highest β-cell function, followed by the Malays  and the OAs . In pre-diabetes, the Chinese had a β-cell function of more than four times that of the Malays and the OAs. There was no significant difference in β-cell function of diabetic Malay, Chinese, Indian and OA subjects.Significant differences can be seen in the β-cell function of Malay, Chinese, Indian and OA subjects in non-diabetes (p = 0.001). In diabetic subjects, Malays  had almost double the RAGE concentration compared to Chinese  and Indian . We could not obtain comprehensive measurement of serum RAGE concentration in the OA due to a technical problem with the ELISA kit.There was no significant difference in the serum RAGE concentration in different ethnicities who were non-diabetic or pre-diabetic. However, a significant difference was seen in serum RAGE concentration in Malay, Chinese, Indian and OA subjects with diabetes . High insulin resistance  was correlated with a more than two times higher likelihood of having pre-diabetes compared to non-diabetes.The low corrected serum α-tocopherol concentration was correlated strongly with pre-diabetics, compared to non-diabetics, with more than four times the likelihood . A high serum RAGE concentration was linked with a more than three times higher likelihood  of having diabetes compared to pre-diabetes.Low β-cell function was correlated strongly with diabetics compared to pre-diabetics, with more than five times the likelihood  of diabetes compared to non-diabetes. High insulin resistance was correlated with almost two times the likelihood  of diabetes compared to non-diabetes. Low β-cell function  and high serum RAGE concentration  were linked to diabetes, compared to non-diabetes, with three times higher likelihood.A low corrected serum α-tocopherol concentration was linked with almost three times the likelihood , β-cell function  or serum RAGE concentration .p = 0.185). However, when the serum α-tocopherol concentration was corrected based on the corresponding lipid levels of the study subjects, a significant difference was seen (p = 0.000002). This might be because vitamin E is lipophilic and is transported in the blood with cholesterols and triglycerides [In the present study, there was no significant difference in uncorrected serum α-tocopherol concentration between the non-diabetics, pre-diabetics and diabetics (ycerides . The lipp < 0.001). Many previous studies, such as the Eastern Finland study [p < 0.001), the data also show that it was specifically implicated in an earlier stage, in pre-diabetes. This contrasts with previous studies, which only correlated it with the development of frank diabetes. From these findings, we speculate that a low corrected serum α-tocopherol concentration is a risk factor in the development of pre-diabetes. A main difference between the study population of the Eastern Finnish study and the current study is that their study population had a significantly lower α-tocopherol concentration (mean: 19 μmol/L) compared to our study population (mean: 41 μmol/L). Although the mechanism is not known, it is possible that the difference in α-tocopherol concentration between the two populations leads to the differences seen in the risk profiles of development of type 2 diabetes.The lowest corrected serum α-tocopherol concentration was found in the pre-diabetic stage. In diabetes, the corrected serum α-tocopherol concentration was also lower than non-diabetes. This crucial information shows that a lower than normal (non-diabetes) corrected serum α-tocopherol concentration is implicated in the development of diabetes, especially in the stage of pre-diabetes. Moreover, the data from this study show that a low corrected serum α-tocopherol concentration was correlated with a more than four times likelihood of having pre-diabetes compared to non-diabetes  in diabetic subjects in comparison with non-diabetic subjects. Additionally, the β-cell function in diabetic subjects was half that of non-diabetics . Study subjects with high insulin resistance  and low β-cell function  also had a higher likelihood of being diabetic than being non-diabetic. These findings are in keeping with previous findings that a combination of high insulin resistance and low β-cell function are key factors in the development of type 2 diabetes.Increasing insulin resistance and decreasing β-cell function are hallmark events that lead to the development of impaired glucose tolerance and, eventually, type 2 diabetes . Data frp < 0.05) compared to the non-diabetics and diabetics. Inversely, β-cell function was at the highest in our pre-diabetic subjects . This means that in our study population, β-cell compensation can still be seen in pre-diabetes. As suggested by a previous study, β-cell function has to be assessed in correlation with insulin resistance [A closer examination of the relationship of insulin resistance and β-cell dysfunction with the stages of development of type 2 diabetes yields valuable information. Pre-diabetic study subjects in the current study were characterized by having the highest insulin resistance  were linked with a more than twofold likelihood of having pre-diabetes compared to non-diabetes. On the other hand, subjects with low β-cell function have six times higher likelihoods of having diabetes compared to pre-diabetes . These findings lead us to postulate that in Asians, high insulin resistance is an important risk factor in the development of pre-diabetes from non-diabetes, while low β-cell function is more crucial in the development of diabetes from pre-diabetes. To support our hypothesis, a recent study in China similarly found that insulin resistance and β-cell dysfunction are important determinants in the development of pre-diabetes and diabetes, respectively [The current study also looked at odds ratios of insulin resistance and β-cell function in non-diabetes, pre-diabetes and diabetes to quantify the risk factors. Data from the current study found that subjects with high insulin resistance . In contrast, diabetics had a 25% higher median serum RAGE concentration compared to pre-diabetics . Data from the current study also found that subjects with high serum RAGE concentration were three times more likely to have diabetes compared to pre-diabetes . The key question now is whether a high serum RAGE concentration is a risk factor for the development of diabetes from pre-diabetes due to RAGE’s effect on β-cell function, or whether a high serum RAGE concentration is a direct consequence of chronic hyperglycemia from the development of frank diabetes. Although we can see that a high serum RAGE concentration is linked to the development of diabetes from pre-diabetes, more studies need to be conducted to understand the mechanism behind this relationship.In the current study, there was a modest 2% higher median serum RAGE concentration in pre-diabetics compared to non-diabetics  have the lowest corrected serum α-tocopherol concentration compared to the Chinese  and the OAs . The α-tocopherol level in pre-diabetic Malays is also 12% lower (0.6948 vs. 0.7874) than the overall population. This trait might pose an additional risk of diabetes, as having the lowest corrected serum α-tocopherol concentration is shown to be a risk factor of pre-diabetes and diabetes. The majority of the subjects in the present study did not take vitamin E supplements. As such, it is possible that this variation in α-tocopherol concentration could be due to differences in lifestyle and dietary intake, or due to genetic variation leading to differences in the metabolism of the antioxidant.Based on the data of the current study, in pre-diabetes, the Malays  and pre-diabetes (p < 0.001). Additionally, their insulin resistance increases steadily from non-diabetes to pre-diabetes and to diabetes, a stark difference compared to the trend seen in the overall population, where insulin resistance peaks in pre-diabetes. Considering high insulin resistance as a risk factor of pre-diabetes, this is a favorable trait for the Malays. The Malays have significantly lower insulin resistance compared to the overall population across all diabetic stages. Correspondingly, the Malays’ β-cell function is significantly lower than other ethnicities’ in terms of non-diabetes (p = 0.001). In diabetes, the Malays (5579.31 pg/mL) have almost double the serum RAGE concentration of the Chinese (3473.40 pg/mL) and of the Indians (3279.52 pg/mL). Additionally, no significant difference is found in serum RAGE concentration across the diabetic stages in the Chinese and Indians. The RAGE concentration of the OAs could not be obtained due to a technical problem with the ELISA kit. Due to their high serum RAGE concentration, the Malays may have a higher risk of diabetes due to the β-cell toxicity of RAGE. These findings regarding RAGE levels may also suggest Malays could be at a higher risk of diabetic complications as the AGE–RAGE interaction can lead to endothelial dysfunction due to increased inflammation and oxidative stress.On the other hand, although the trends of increase in serum RAGE concentration are similar between the Malays and the overall study population, the degree of increase in serum RAGE concentration between pre-diabetes and diabetes is much higher in Malays (~700%) compared to the study population (~125%). It is also important to note that there are no significant differences seen in serum RAGE concentration in other ethnicities as diabetes progresses from non-diabetes to pre-diabetes and eventually diabetes. The current study also finds a significant difference in serum RAGE concentration between ethnic groups who are diabetic (p = 0.023) compared to the other ethnicities. The corrected serum α-tocopherol concentration of pre-diabetic Chinese was also 8% higher (0.8564 vs. 0.7874) than the overall study population. This higher α-tocopherol level could be a protective factor against pre-diabetes for the Chinese.In the present study, among the pre-diabetics, the Chinese had the second highest corrected serum α-tocopherol concentration  and pre-diabetics . Compared to the overall study population, their insulin resistance was 55% higher in non-diabetics (0.0174 vs. 0.0112) and 65% higher (0.0357 vs. 0.0217) in pre-diabetics. As a result, the β-cell function of the Chinese is correspondingly high in non-diabetes  and in pre-diabetes , compared to the other ethnicities. As discovered in the current study, having higher insulin resistance puts the Chinese at a higher risk of the development of diabetes from the pre-diabetic stage.Data from the present study show that the Chinese had significantly higher insulin resistance compared to the other ethnicities in non-diabetics , and correspondingly, the second highest β-cell function  compared to the other ethnicities. However, when compared to the overall study population, the data showed that the Indians’ insulin resistance (0.0113 vs. 0.0112) and β-cell function (5.00 vs. 4.15) were similar to the overall. This might indicate that the Indians have an average risk profile compared to the overall population in terms of insulin resistance and β-cell function. A comparable sample size could not be obtained for the pre-diabetic Indians.In non-diabetics, the Indians in the current study had the second highest insulin resistance . The corrected serum α-tocopherol concentration of pre-diabetic OAs was 13% higher (0.8938 vs. 0.7874) compared to the overall study population. As such, like the Chinese, the higher α-tocopherol level could protect the pre-diabetic OAs against the progression to diabetes.In the current study, the pre-diabetic OAs had the highest corrected serum α-tocopherol concentration  and pre-diabetes . Additionally, in comparison with the overall study population, the OAs’ insulin resistance was lower in non-diabetes (0.0083 vs. 0.0112) and pre-diabetes (0.0111 vs. 0.0217). Correspondingly, the OAs’ β-cell function was low in non-diabetes  and pre-diabetes , compared to the other ethnicities. There were also no significant differences in the OAs’ insulin resistance and β-cell function across diabetic stages, as they remained low. The low insulin resistance can be a protective trait against diabetes for the OAs.Data from the present study found that the OAs had lower insulin resistance in comparison with other ethnicities in non-diabetes (0.0083; These examples show the potential of an individualized therapy at different stages of diabetes based on the risk factor each person exhibits, when the biomarkers of the diabetes risk factors tested in this study or newer biomarkers become more accessible. Notably, in diabetes, the ethnical differences in corrected α-tocopherol concentration, insulin resistance and β-cell function seen in non-diabetes and pre-diabetes were not significant. Moreover, a significant difference in corrected α-tocopherol concentration amongst multiple ethnicities was seen only in pre-diabetes. As such, secondary prevention efforts for individuals with pre-diabetes could address this and close the ethnical gap in diabetic risk factors. In other words, this makes pre-diabetes a suitable phase for interventional efforts, such as vitamin E supplementation, to improve the α-tocopherol levels as well as to better the insulin resistance and β-cell function. In addition, the long duration of pre-diabetes of up to 8–10 years  providesMalaysia is a multiethnic country, as each ethnic group has its own unique culture and cuisine. The multiethnic people consume a vast variety of diets comprising different compositions of macronutrients and micronutrients, as well as antioxidants, such as natural sources of α-tocopherol. This environmental factor, unique to this country, could to some degree contribute to the difference in the risk profiles seen. Additionally, the disparity in access to healthcare facilities and in education level can give rise to different degrees of diabetic education and diabetic treatment. This could also bring about the difference in the diabetes risk factors studied, such as insulin resistance, β cell function and serum RAGE levels. Other than that, it is increasingly recognized that age, hyperglycemia  and obesAll in all, the differences in corrected α-tocopherol concentration, insulin resistance, β-cell function and serum RAGE concentration in subjects of different ethnicities gives rise to different risk profiles of diabetes. As diabetes is a multifactorial disease, these factors contribute to different degrees to the differences in diabetes prevalence seen in national surveys, along with other genetic and lifestyle factors. The current study found that the OAs had the highest corrected serum α-tocopherol concentration in the pre-diabetics, followed by the Chinese and the Malays. The Chinese have the highest while the Malays have the lowest insulin resistance and β-cell function, compared to the other ethnicities in the non-diabetics and pre-diabetics. The diabetic Malays have significantly higher serum RAGE concentrations compared to the non-diabetic and pre-diabetic Malays, as well as in comparison with other ethnicities. The differential risk profiles seen in different ethnicities can give rise to the ethnical variation in diabetes prevalence seen in national surveys to different degrees along with other genetic and environmental risk factors, as diabetes is a multifactorial disease.The ethnical variations in the corrected α-tocopherol concentration, insulin resistance and β-cell function seen in non-diabetic and pre-diabetic subjects were not significant in the diabetic subjects. This signifies that an interventional effort should be made in the pre-diabetic stage to close the ethnical gaps in risk factors, before it is too late.We also postulate that a low corrected serum α-tocopherol concentration and a high insulin resistance are key factors in the development of pre-diabetes. Conversely, low β-cell function and high serum RAGE concentration are linked strongly to the development of diabetes. Prospective cohort studies should be done in the future to investigate causality more precisely.


Purpose: Skin-derived Precursor Schwann cells (SKP-SCs) have been reported to provide neuroprotection for the injured and dysmyelinated nervous system. However, little is known about SKP-SCs on acute ischemic stroke (AIS). We aimed to explore the efficacy and the potential mechanism of action of SKP-SCs on AIS in a rat ischemic stroke model.Methods: Adult male Sprague–Dawley rats were subjected to a middle cerebral artery occlusion (MCAO) for 1.5 h on Day 0 and subsequently received an intracarotid injection of 2 × 106 green fluorescent protein (GFP) -labeled SKP-SCs or phosphate buffered saline (PBS) during reperfusion. Neurological function was assessed by behavioral tests on Days 1, 4, 7, 14, and 28. In a satellite cohort, rat brains were harvested and infarct volume was measured with 2,3,5-triphenyltetrazolium chloride (TTC) staining on Days 1 and 7, and migration and survival of SKP-SCs in the brain were traced by monitoring green fluorescence at 6 and12 h on Day 0, and on Days 1, 4, 7, 14, and 28. Histopathology and immunofluorescence staining were used to analyze the morphology, survival and apoptosis of neurons. Additionally, in an in vitro SKP-SC co-culture model using fetal rat primary cortical neurons underwent oxygen glucose deprivation/reoxygenation (OGD/R), Western blot was used to detect the expression of apoptosis indicators including activated caspase-3, Bax, and Bcl-2. TUNEL staining was used to count apoptotic cells.Results: Intracarotid transplantation of SKP-SCs effectively migrated to the periinfarct area and survived for at least 4 weeks. Transplanted SKP-SCs inhibited neuronal apoptosis, reduced infarct volume, and improved neurological recovery in the MCAO rats. Moreover, in vitro data showed that SKP-SCs treatment inhibited OGD/R-induced neuronal apoptosis and promoted survival of the cultured primary cortical neurons.Conclusions: Intracarotid transplantation of SKP-SCs promoted functional recovery in the rat AIS model and possesses the potential to be further developed as a novel therapy to treat ischemic stroke in humans. Stroke is the leading cause of neurological disability in adults worldwide , with thStudies have shown that recovery/compensation of neural functions after stroke could actually last for months, depending on the plastic environment of the ischemic brain and the degree of neural circuit remodeling . This prSkin-derived precursors (SKPs) are dermally derived pluripotent precursor cells that are isolated and cultured from murine dorsal skin or human foreskin –8. TheseWe hereby hypothesize that SKP-SCs will have therapeutic effect on ischemic stroke by improving the disruption of neural circuitry and suppressing ischemia-induced apoptosis in the infarcted brain region. To test this hypothesis, we have transplanted SKP-SCs via a novel intracarotid route during reperfusion in a transient rat middle cerebral artery occlusion (MCAO) model. We found that SKP-SCs inhibited apoptosis, reduced infarct size, and improved neural functional recovery in behavioral test.ad libitum. After at least 3 days habituation, rats were randomly assigned into three groups: sham operation, MCAO + PBS and MCAO + SKP-SCs. The sample size estimation for behavioral studies was based on the effects of SKP-SCs in our previous experiment . Standard biosafety and the institutional standard operating procedures were strictly followed in SKP-SCs and all cell culture experiments. Healthy male Sprague-Dawley (SD) rats were housed in cages on a 12-h light/dark cycle. Food and water were available periment . A samplTransient MCAO was induced by intraluminal vascular occlusion surgery following the method previously described . Briefly6 cells) was carefully infused through the tube for 10 min in MCAO + SKP-SCs group. This dose was selected based on the results of previous studies . Two investigators designated for the test were blinded to both the surgery and transplantation. Sensorimotor deficits were evaluated by improved neurological deficit score (mNSS) and rotarod. Weight changes in rats were also recorded.mNSS is an 18-point scoring system that includes movement , sensation , reflex  and balance assessments. Higher scores indicate more severe neurological damage.Motor coordination and balance were assessed by rotarod test. The fatigue rotator  accelerates from 4 to 40 rpm in 5 min. The length of time the rats remained on the smooth rotating rod was measured. Rats were trained for a consecutive 3 days before MCAO procedures to adapt to the rod-rotator. The maximal exercise time for each training/test was 300 s. Only rats that were able to stay on rotarod for 290–300 s were included in the experiment. The results acquired 1 day before surgery was recorded as baseline. At the time point of postoperative behavioral test, three measurements with an interval of more than 5 min were recorded, and the average was calculated.Baseline body weight of rats was measured and recorded 1 day before surgery, and rats with the weight of 190–220 g were included in the experiment. Body weight of each rat was recorded before behavioral tests were performed at each time point.total brain volume of the intact left  hemisphere minus the leftover healthy brain volume of the right  hemisphere, divided by the total volume of the left hemisphere).Rats from a satellite cohort were sacrificed 1 day and 7 days after SKP-SCs transplantation, and cerebral infarction volume was measured after 2,3,5-triphenyltetrazolium chloride (TTC) staining. Briefly, after deep anesthesia, rats were transcardially perfused with saline. The brains were quickly removed, and the coronal slices cut at 2 mm with a rat brain mold . These slices were immediately stained with 2% TTC solution  in PBS for 30 min in a 37°C incubator. Normal brain tissue is stained red, while infarcted areas remain unstained. The size of the infarcted area was measured by Image J software  and the infarct volume is expressed as the percentage of the infarcted brain volume in the lesioned  hemisphere over the intact left, contralateral hemisphere  and xylazine  (i.p.) and perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer following a quick 0.9% NaCl saline flush. After perfusion, the brains were quickly removed and post-fixed for 24 h in the same fixative, followed by immersion in 20 and 30% sucrose. Coronal brain sections were cut at 12 μm with a Leica cryostat and mounted on slides precoated with adhesive. After drying at room temperature, the slides were boxed and stored at−20°C before use.Histopathologic changes of the cortex and striatum in the penumbra area were studied using H&E  staining in rats of Day 4. After fixation, the sections were stained in hematoxylin solution for 30 s and eosin solution for 1 min. Following gradient dehydration, the slides were further cleared with xylene and coverslipped with neutral gum. The number and morphology of neurons were examined under microscope.Nissl staining was used in rats of Day 4 to further observe pathologic changes of neurons in the penumbra. After being thawed to room temperature, slides were rinsed with distilled water for 5 min to remove the gel around the tissue. Staining was achieved by immersing the slides in the Nissl Staining Solution  for 10–20 min at room temperature. After brief rinsing, all slides went through ascending dehydration and were cleared in xylene before being coverslipped.Tissues of MCAO+ SKP-SCs group were directly observed under a fluorescence microscope  to detect survival and migration of GFP-labeled SKP-SCs in the rat brain at 6 h, 12 h on Day 0, Days 1, 4, 7, 14 and 28 after MCAO. To further observe the health of neurons and glial cells, as well as neuronal dendritic processes and axonal growth, additional sets of slides were stained with antibodies against different neuronal and glial cell markers, including NeuN, GFAP, MAP2, and GAP43. After temperature equilibration and drying, sections mounted on slides were washed with PBS for 10 min, then blocked with 0.1% Triton x-100 for 2 h, before being incubated overnight at 4°C with the following antibodies: rabbit anti-NeuN antibody  and rabbit anti-GFAP antibody , mouse anti-MAP2 antibody , and rabbit anti-GAP43 antibody. Indirect fluorescence by incubating sections at room temperature for 2 h with goat-anti-rabbit IgG conjugated with Alexa Fluor 594 or goat-anti-mouse-IgG conjugated with Alexa Fluor 488  was used for detection. Sections were further incubated with Hoechst for 10 min before being coverslipped for microscopic observation.TUNEL assay was used to examine the apoptosis in the penumbra. After taken out of box, slides were dried at 37°C for 1 to 2 h and rinsed with PBS before being post-fixed with 4% paraformaldehyde for 30 min. Following block with 1% Triton X-100 for 10 min, 50 μl of TUNEL test solution  was applied to each slide and incubated for 1 h at room temperature. After that, slides were further counterstained with Hoechst for 5 min. Apoptosis index was calculated by counting the total number of TUNEL positive cells and all the number of Hoechst positive cells.in vivo study design is illustrated in The whole in vitro co-culture experiment using rat primary cortical neurons (PCNs) and SKP-SCs was designed and performed.To explore the mechanism of action that SKP-SCs salvage injured neurons in the penumbra region after ischemia, an 4 in a 24-well plate, 1 × 105 in a 96-well plate, and 3 × 106 in a medium dish. Once cells adhered to the wall after culture for 4 to 6 h, the culture medium was replaced with the whole culture medium .Timed pregnant SD rats were anesthetized with ether and fetuses (E17) were removed from the uterus. Fetal brains were quickly removed and placed in pre-chilled DMEM medium. Cerebral cortex was isolated and cut it into pieces. After digestion with 0.25% trypsin at 37°C for 7 min, DMEM containing 10% fetal bovine serum was added. Homogenization was achieved by repeated pipetting. The suspension was then centrifuged at 12,000 rpm for 5 min and cells resuspended in DMEM with 10% fetal bovine serum and filtered. Cells were then seeded on cell culture plates and petri dishes, with the inoculation density of 5 × 10in vivo. After replacing Neurobasal medium containing B27, L-glutamine, and antibiotics with glucose-free DMEM medium, the PCNs culture plate was placed in a 37°C hypoxia incubator for 1.5 h. Once the oxygen-glucose deprivation was completed, the cell culture solution was replaced with Neurobasal medium and reoxygenated, and further cultured in a regular cell culture incubator at 37 °C with 20% oxygen and 5% CO2.OGD/R procedures were used to simulate the effect of MCAO and reperfusion on neurons DMEM/F12 FBS-free medium was used to prepare SKP-SC CM when SKP-SCs density reached ~80%. After further culture for 48 h, SKP-SCs CM were collected and concentrated using a centrifugal filtration device  at 3000 × g. SKP-SCs-free control medium was prepared in the same way. Concentrated (about 50-fold) CM samples were quickly frozen and stored at −80°C until use.Cell viability was assessed with a Cell Counting Kit-8 assay kit (CCK-8) . PCNs were cultured for 6 days and treated with or without OGD for 1.5 h followed by addition of SKP-SCs CM or control medium for co-culture for another 24 h during reoxygenation. After adding 10 μl of CCK-8 solution to each well, the 96-well plates were incubated in a cell incubator for 1.5 h and the absorbance was measured at 450 nm with a microplate reader .Before adding CCK-8 assay kit, some of the above co-cultured PCNs were harvested. These cells were lysed with Cell Lysis  to obtain whole-cell lysate. BCA protein assay kit  was used to measure protein concentration. Twenty micrograms of protein at the concentration of 4 μg/μl was loaded to each well of SDS-PAGE gel  for electrophoresis, and the separated proteins were transferred to PVDF  membranes. After blocking with 5% skim milk at room temperature for 2 h, the membranes were incubated with the following primary antibody at 4°C overnight: cleaved caspase-3 , Bax , Bcl-2 , and GAPDH . The membranes were further incubated with an HRP-conjugated secondary antibody at room temperature for 2 h and reacted with enhanced chemiluminescence (ECL) agent . Protein signals were detected by an automatic chemiluminescence/fluorescence image analysis system . Quantification was performed using Image J software .t test was used for pairwise comparisons. A two-sided one-way ANOVA and post hoc Bonferroni test were used to compare multiple groups. A value of p < 0.05 was considered statistically significant.GraphPad Prism 5.0  software was used for statistical data analysis. All data were presented as mean ± SEM. Student's Schwann cell phenotype of the SKP-SCs was characterized before transplanting. SKP-SCs showed typical bipolar morphology of Schwann cells . ImmunofSKP-SCs were clearly detected in the ischemic region of the right cerebral hemisphere as early as 6 h after transplantation, although some SKP-SCs remained in the blood vessels , 6 h. Thp < 0.01), accounting for a 22.87% reduction (TTC staining demonstrated a remarkable infarction in the right hemisphere . SKP-SCseduction .p < 0.001). In contrast, PBS control displayed no effect (p < 0.05 ~ 0.001) (p < 0.001)  . The mea< 0.001) . CollectThe neuronal survival and morphological changes in the penumbra region were analyzed with H&E staining and Nissl staining on Day 4. Comparing with rats with sham surgery , Sham, Mp < 0.001)  .p < 0.05). The viability of SKP-SCs CM cultured PCNs appeared to be comparable to that of PCNs without any oxygen/glucose deficiency (p < 0.001)  . However< 0.001) .p < 0.001). This increase, however. was remarkably inhibited by the addition of SKP-SCs CM  (p < 0.05) (The number of TUNEL-positive cells was markedly reduced by SKP-SCs CM compared with the control . Proteind Cas-3) . On the  < 0.05) .in vivo MCAO model and an in vitro primary cortical neuron culture with OGD/R model were used to explore whether SKP-SCs cell therapy promotes neuroprotection after AIS. It was found that SKP-SCs can survive and maintain their phenotype up to 4 weeks. Intracarotid transplantation of SKP-SCs effectively migrated to the periinfarct area, inhibited neuronal apoptosis, reduced infarct volume, and improved functional recovery in the MCAO rats. Moreover, SKP-SCs CM inhibited OGD/R-induced neuronal apoptosis and promoted neuronal survival in the peri dish. Given the favored extended time window, our study has provided the first evidence that SKP-SCs could be a potential novel cell therapy for patients with AIS, in addition to the current standard of care.SKP-SCs have been previously shown to promote repair and functional recovery in rats with spinal cord injury , 13, 18.The delivery route and the optimal time point for SKP-SCs treatment are two of the important issues that must be addressed in future clinical development. As described in previous studies , many dein vivo is of vital importance , analyzed data, prepared the figures, and wrote the manuscript. RC performed the experiments  and contributed to the analysis and interpretation of data. JW performed the experiments . KK designed the study and critically reviewed the manuscript. JS, YC, and MC supervised the project and revised the manuscript critically. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.